E-ISSN 2549-8703 I P-ISSN 2302-7282 BIOTROPIKA Journal of Tropical Biology https://biotropika.ub.ac.id/ Vol. 9 | No. 2 | 2021 | DOI: 10.21776/ub.biotropika.2021.009.02.09 GENETIC DIVERSITY OF Dacrycarpus imbricatus AT BUKIT TAPAK, TABANAN, BALI BASED ON RAPD MARKER KERAGAMAN GENETIK Dacrycarpus imbricatus DI BUKIT TAPAK, TABANAN, BALI BERDASARKAN PENANDA RAPD Made Pharmawati 1)*, I Made Anom Sutrisna Wijaya2) Received : May, 29 2021 ABSTRACT Dacrycarpus imbricatus Blume is a member of the Podocarpaceae family. In Bali, D. Accepted : July, 13 2021 imbricatus was found in Bukit Tapak, Tabanan Regency. This species is one of the dominant species in Bukit Tapak. This study aimed to determine the genetic variation of D. imbricatus in Bukit Tapak using molecular markers RAPD (Random Amplified Polymorphic DNA). The genetic diversity of D. imbricatus needs to be studied to obtain Authors affiliation: the information used for the conservation of this species. Leaf samples were taken from 1) Biology Department, Faculty of Bukit Tapak, Candikuning, Baturiti, Tabanan Regency, Bali. DNA was extracted using Mathematics and Natural the CTAB method, followed by extraction using chloroform: isoamyl alcohol. DNA Sciences, Universitas Udayana, precipitation was carried out using ethanol. RAPD analysis was performed using Kampus Bukit Jimbaran, Bali polymerase chain reaction (PCR) using four primers. PCR products were visualized 2) Department of Agricultural and using agarose gel electrophoresis and ethidium bromide staining. The results showed that Biosystems Engineering, the amplified DNA bands ranged from 1 to 5 bands with DNA band sizes ranging from Faculty of Agricultural 230 bp - 422 bp. Only OPA4 and UBC106 primers can be used to detect D. imbricatus Technology, Universitas diversity based on the H, D, and R values. The detected genetic variation is low, as Udayana, Kampus Bukit indicated by an average polymorphism of 32.5% and similarities between samples Jimbaran, Bali ranging from 0.51 to 1. Keywords: Bukit Tapak, Dacrycarpus imbricatus, polymorphism, RAPD Correspondence email: ABSTRAK *[email protected] Dacrycarpus imbricatus Blume adalah anggota dari famili Podocarpaceae. Di Bali, D. imbricatus ditemukan di Bukit Tapak, Kabupaten Tabanan. Spesies ini merupakan salah satu spesies yang dominan di Bukit Tapak. Penelitian ini bertujuan untuk menentukan variasi genetik D. imbricatus di Bukit Tapak menggunakan penanda molekuler RAPD (Random Amplified Polymorphic DNA). Keragaman genetik D. imbricatus perlu dipelajari untuk mendapatkan informasi yang digunakan untuk konservasi spesies ini. Sampel daun diambil dari Bukit Tapak, Candikuning, Baturiti, Kabupaten Tabanan, Bali. Ekstraksi DNA menggunakan metode CTAB dilanjutkan dengan menggunakan kloroform: isoamylalkohol. Pengendapan DNA dilakukan menggunakan etanol. Analisis RAPD dilakukan dengan menggunakan polymerase chain reaction menggunakan empat primer. Produk PCR diamati menggunakan elektroforesis gel agarosa dan pewarnaan How to cite: etidium bromida. Hasil penelitian menunjukkan bahwa pita DNA yang teramplifikasi Pharmawati, M, IMAS Wijaya. berkisar 1 – 5 pita dengan ukuran pita DNA berkisar 230 bp – 422 bp. Keragaman D. 2021. Genetic diversity of imbricatus hanya terdeteksi bila menggunakan primer OPA4 dan UBC106 berdasarkan nilai H, D dan H. Variasi genetik yang terdeteksi tergolong rendah yang ditunjukkan Dacrycarpus imbricatus at Bukit dengan rata-rata polimorfisme 32,5% dan similarity antar sampel berkisar 0.51 sampai Tapak, Tabanan, Bali based on 1. RAPD marker. Journal of Tropical Biology 9 (2): 157-161. Kata kunci: Bukit Tapak, Dacrycarpus imbricatus, polimorfisme, RAPD INTRODUCTION In Bali, the habitat of D. imbricatus is in Bukit Tapak, Tabanan Regency. Bukit Tapak Forest is Dacrycarpus imbricatus Blume or also known located at an altitude of 1520-1600 m asl [2]. This as Podocarpus imbricatus is a member of plant in Bali is known as the local name cemara Gymnosperms and belongs to the Podocarpaceae pandak. D. imbricatus is one of the dominant family. This plant is a native species of Indonesia species in Bukit Tapak. Bukit Tapak forest is and is distributed in Sumatra, Java, Sulawesi, classified as a secondary forest [3]. D. imbricatus Lesser Sunda, and Papua. This species grows in the is a long-lived pioneer species that grew due to past highlands from 500 to 2,000 m asl (above sea level) disturbances in its territory [4]. Conservation of D. [1]. imbricatus is needed to prevent the loss of this species in the area due to species change. Pharmawati & Wijaya 157 https://biotropika.ub.ac.id/ Information on the genetic diversity of D. imbricatus is important for developing conservation strategies. Preservation of genetic diversity is necessary for the survival of a species over a long period. Loss of genetic diversity will decrease the ability to adapt to environmental changes [5]. Genetic diversity studies can identify alleles that can contribute to an organism's ability to survive in its habitat or survive in a varied habitat. Loss of genetic diversity can lead to extinction in small populations due to decreased environmental adaptability [6, 7]. The detection of genetic diversity can be carried out by several methods. The genetic diversity of plants has traditionally been observed based on Figure 2. D. imbricatus tree in Bukit Tapak morphological characters or growth responses. In this method, individuals are differentiated based on DNA extraction. The leaves were washed with their phenotypic character, which can be soap and running water until clean, then 0.1 g of influenced by the environment [8]. The leaves were crushed using a mortar and pestle. As development of molecular markers provides a much as 1 ml of CTAB buffer (2% w / v CTAB, sensitive method for detecting diversity at the DNA 1.4 M NaCl, 50 mM EDTA, 100 mM Tris-HCl (pH level. One of the techniques used in DNA 8), 2% v / v 2- mercaptoethanol) was added [10, fingerprinting in evaluating genetic diversity is 11]. Incubation of the mixture was done at 65ºC for RAPD (Random Amplified Polymorphic DNA). 1.5 hours. Following incubation, centrifugation The RAPD technique detects DNA polymorphisms was done at 14,000 rpm for 5 minutes. using single primers with random sequences [9]. The supernatant was collected to a new tube and RAPD analysis is useful for detecting nucleotide added 500 µl of chloroform: isoamyl alcohol (24: polymorphisms that show genetic diversity. This 1). The mixture was vortexed and centrifuged for 5 study is aimed to analyze the genetic variation of minutes at 14,000 rpm. The top layer was removed D. imbricatus in Bukit Tapak, Tabanan, Bali by and transferred to a new tube, and purification using RAPD. The results of this study can be used using chloroform: isoamyl alcohol (24:1) was as a basis for developing a conservation strategy. repeated. The top layer was transferred to a new tube, and 500 µl of chilled ethanol was added and METHODS incubated overnight. After that, the sample was Sampling of plant materials. D. imbricatus centrifuged at 8,000 rpm for five minutes. The leaves were taken from Bukit Tapak, Tabanan pellets were washed using 70% ethanol and Regency, Bali (Figure 1). The leaves used were centrifuged for 5 minutes at 8,000 rpm. The pellets fully developed leaves. The samples used were 16 were air-dried and resuspended in 100 µl of sterile individuals’ trees of D. imbricatus (Figure 2). H2O [10, 11]. DNA was visualized with agarose gel (1%) electrophoresis in TAE buffer for 45 minutes at 100 volts. DNA staining was done with ethidium bromide. Visualization was performed using UV- transilluminator [12]. The DNA concentration was estimated by comparing with 50 ng and 100 ng of lambda () DNA. RAPD. PCR-RAPD was conducted in 20 µl volume with 25 ng DNA, 1 x PCR buffer, 200 µM dNTP, 3 mM MgCl2, 1 mM primer, and 1 unit taq polymerase. Eight primers from operon primers (OP) and the University of British Columbia (UBC) were screened (Table 1). Figure 1. Map of the location of Bukit Tapak (arrow) based on Google Earth 158 Biotropika: Journal of Tropical Biology | Vol. 9 No. 2 | 2021 https://biotropika.ub.ac.id/ Table 1. Name of primers screened and their cells are exposed to lysis buffer (CTAB) longer so sequences that a better DNA lysis process occurred [17]. The EDTA concentration in the CTAB lysis Primer Name Sequence (5’-3’) buffer used in this study was also higher than that OPA2 TGCCGAGCTG of Doyle & Doyle [10]. Modifications have been OPA4 AATCGGGCTG made previously using 50 mM EDTA [11]. In the OPB8 GTCCACACGG lysis buffer, EDTA functions as a chelator of Mg2+, OPD11 AGCGCCATTG where Mg2+ is a cofactor of the enzyme nuclease OPD14 CTTCCCCAAG [18]. By increasing the EDTA concentration, it is OPH1 GGTCGGAGAA expected that the amount of Mg2+ was chelated UBC106 CGTCTGCCCG more, thereby inhibiting nuclease activity. UBC250 CGACAGTCCC Diversity analysis with RAPD. DNA amplification by PCR-RAPD was successfully The PCR cycle was 1x initial denaturation at performed using four primers (OPA4, OPD14, 95ºC for 5 minutes, 40 x denaturation at 95ºC for 1 UBC106, and UBC250) of the eight primers tested. minute, primer annealing at 37oC for 1 minute, Only the four primers mentioned above produced extension at 72ºC for 1 minute. The final extension clear PCR products, while the other four primers was carried out for 10 minutes at 72ºC. PCR (OPA2, OPB8, OPD11, and OPH1) produced products were visualized using 1.8% agarose gel products with a very strong background smear so electrophoresis with ethidium bromide staining. that the DNA bands were not clearly observed. Data analysis. Existing DNA bands were score Figure 4. shows the PCR-RAPD results using UBC 1, whereas the absence of DNA band was scored 0. 106 primers with a 1kb ladder as a size marker. Primary efficiency analysis was performed using Primer selection in diversity analysis plays an iMEC [13]. The kinship between individuals was important role. The analysis of the RAPD primer analyzed using NTSYS 2.1 [14], with the UPGMA efficiency is shown in Table 2, while the number method with a simple matrix similarity coefficient.